PROJECT SUMMARY Bacteria have evolved specialized nanomachines to deliver microbial cargo across the cell envelope. One versatile translocation apparatus, the type IV secretion system (T4SS), can be strategically deployed to inject macromolecular substrates into target bacterial or eukaryotic recipient cells. Despite their importance in bacterial pathogenesis and dissemination of antibiotic resistance determinants, the mechanisms by which the T4SS assembles and transports payload remain largely undefined. To address this knowledge gap, the long-term goal of this proposal is to develop and apply robust molecular tools to accelerate fundamental studies of T4SS nanomachines. The cag T4SS of the gastric bacterium Helicobacter pylori has emerged as an important system for understanding how a single molecular machine can transport diverse cargo into target cells. Whereas some T4SS have the capacity to secrete hundreds of proteins or DNA-protein complexes into the host cell, the ability to translocate a diverse repertoire of lipid, nucleic acid, protein, and polysaccharide substrates distinguishes the cag T4SS from other systems. Notably, the bacterial oncoprotein CagA is rapidly delivered to host gastric cells via cag T4SS mechanisms. Translocated H. pylori effector molecules activate innate defenses and dysregulate signaling pathways that influence progression of gastric disease; consequently, colonization by cag T4SS- positive H. pylori significantly augments the risk for gastric cancer. As a result of its central role in bacterial pathogenesis, the T4SS represents an ideal target for antimicrobials. In this application, we propose to identify and mechanistically characterize novel small molecule-based T4SS modulators. Iterative structure-activity relationship studies will be used to develop chemical scaffolds and pharmacophores with optimized anti-virulence potential. Probe development will take advantage of expertise and assay platforms in the Shaffer lab and will leverage synergistic resources in the proposed CPRI Computational Core (ligand-binding model development, rational design, virtual screening), CPRI Translational Core (high throughput assay support, novel compound repositories, and ADMET profiling), and the Organic Synthesis Core (medicinal chemistry and scale-up). Prioritized and validated chemical probes will be used in conjunction with biochemical and genetic approaches to interrogate cag T4SS regulation and dynamic steps in substrate translocation. Using a similar multidisciplinary approach, we will determine how the H. pylori cag T4SS apparatus assembles at the bacteria-host cell interface. Collectively, these studies will stimulate new basic research directions and will provide important insight into how the T4SS nanomachine orchestrates the delivery of specific molecular cargo to target cells to drive microbial pathogenesis. Furthermore, this work will generate powerful chemical tools that are broadly applicable to infectious disease research, and will identify potent lead compounds with the potential to disarm T4SS function in a variety of medically-relevant pathogens.